Heparan sulfate proteoglycans (HSPG) are important structural and functional components of basement membranes and are particularly important in the glomerular basement membrane where they play an important role in the selective filtration process. The HSPG are synthesized by epithelial cells and must be secreted in a polarized fashion from the basal surface of the cell for incorporation into the basement membrane. From our previous studies we know that in polarized epithelial cells the basal secretion of the HSPG is accomplished by a sorting step at the level of the trans-Golgi and that the sorting mechanism is pH dependent and relies on interaction with the core protein and not the heparan sulfate chains. The polarized secretion of the HSPG and other molecules in these cells is dependent upon selective and vectorial movement of vesicles between cell compartments and the cell surface. The vesicles transporting the HSPG through the cell must have regulatory proteins associated with their membranes to at as address labels to ensure delivery of the HSPG to the basal cell surface. Recent studies have implicated families of G proteins as regulators in vesicle trafficking. We have developed and will use an established model of HSPG secretion in polarized epithelial cell cultures to study the role of G proteins in the constitutive secretion of basement membrane HSPG. These studies are made feasible by the availability of antibodies to various G proteins which will be used to immunolocalize these proteins and by the availability of cell lines stably transfected with specific G protein genes in which we can study the functional role of these G proteins in secretion. In addition we will take advantage of techniques for "capturing" HSPG- proposed studies will identify G proteins that are present on vesicles transporting HSPG to the basal cell surface and the functional studies will elucidate the role of these G proteins in regulating the constitutive delivery of molecules to the basement membrane. The long term goal of this research is to understand how kidney epithelial cells assemble and maintain the basement membranes that are crucial for kidney structure and function.